Circulating Tumor DNA Is Prognostic in Intermediate-Risk Rhabdomyosarcoma: A Report From the Children's Oncology Group

Abstract

Purpose: Novel biomarkers are needed to differentiate outcomes in intermediate-risk rhabdomyosarcoma (IR RMS). We sought to evaluate strategies for identifying circulating tumor DNA (ctDNA) in IR RMS and to determine whether ctDNA detection before therapy is associated with outcome.

Patients and methods: Pretreatment serum and tumor samples were available from 124 patients with newly diagnosed IR RMS from the Children's Oncology Group biorepository, including 75 patients with fusion-negative rhabdomyosarcoma (FN-RMS) and 49 with fusion-positive rhabdomyosarcoma (FP-RMS) disease. We used ultralow passage whole-genome sequencing to detect copy number alterations and a new custom sequencing assay, Rhabdo-Seq, to detect rearrangements and single-nucleotide variants.

Results: We found that ultralow passage whole-genome sequencing was a method applicable to ctDNA detection in all patients with FN-RMS and that ctDNA was detectable in 13 of 75 serum samples (17%). However, the use of Rhabdo-Seq in FN-RMS samples also identified single-nucleotide variants, such as MYOD1^L122R, previously associated with prognosis. Identification of pathognomonic translocations between PAX3 or PAX7 and FOXO1 by Rhabdo-Seq was the best method for measuring ctDNA in FP-RMS and detected ctDNA in 27 of 49 cases (55%). Patients with FN-RMS with detectable ctDNA at diagnosis had significantly worse outcomes than patients without detectable ctDNA (event-free survival, 33.3% v 68.9%; P = .0028; overall survival, 33.3% v 83.2%; P < .0001) as did patients with FP-RMS (event-free survival, 37% v 70%; P = .045; overall survival, 39.2% v 75%; P = .023). In multivariable analysis, ctDNA was independently associated with worse prognosis in FN-RMS but not in the smaller FP-RMS cohort.

Conclusion: Our study demonstrates that baseline ctDNA detection is feasible and is prognostic in IR RMS.

FIG 1.

Characterization of FN-RMS genomics and ctDNA detection and quantification. (A) CNAs were identified in all 69 tumor samples by ULP-WGS. (B) SNVs were identified by the Rhabdo-Seq panel in 40 of 57 tumor samples with adequate sequencing coverage. ctDNA was detected in the serum in (C) 17% of the patients by ULP-WGS and (D) 24% of the patients by SNV identification with Rhabdo-Seq. (E) In total, 31% of patients had detectable ctDNA using a combination of ULP-WGS for CNA detection and Rhabdo-Seq panel for SNV detection. (F) Plotted are the percent ctDNA values for FN-RMS samples with detectable ctDNA by ULP-WGS (blue circles) and by Rhabdo-Seq (red circles). ctDNA content estimates are similar in cases where ctDNA is detected by both methods. However, the lack of SNVs in some cases results in ctDNA being unmeasurable by Rhabdo-Seq. In other cases, the lower limit of detection in Rhabdo-Seq allows for identification of very low content of ctDNA in some samples. (G) The plot depicts SNVs in genes (y-axis) targeted by the Rhabdo-Seq panel in patients with FN-RMS (x-axis) who have at least one variant found in either a tumor or serum sample. Open black circles represent variants found in the tumor, and solid red circles represent variants in serum. Circle size reflects allelic fraction of the identified variant. The histogram on top depicts ctDNA levels in serum samples from the patient (indicated on the x-axis) by ULP-WGS (gray squares) or SNV allelic fraction (red bar). When there are two SNVs, only the highest allelic fraction is represented by the red bar. The histogram is partitioned into three sections to help visualize values in cases with very low content of ctDNA. The histogram to the right represents the number of variants identified in the cohort. ^aGenes in bold indicate that a case had evidence of two mutations in that gene (FGFR4 in PASSBH, PIK3CA in PARLFB, and IGF1R in PAUSDR). ^bPatient IDs that are in bold indicate cases without available tumor data. ^cPatient IDs indicate cases without available germline DNA. The five cases with SNVs detected in the serum and not the tumor (PANEPE, PAUPKX, PANDKS, PATYWU, and PATZHV) were analyzed with a patient-matched normal DNA sample. AF, allelic fraction; CNA, copy number alteration; ctDNA, circulating tumor DNA; FN-RMS, fusion-negative rhabdomyosarcoma; SNV, single-nucleotide variant; ULP-WGS, ultralow passage whole-genome sequencing.

FIG 2.

Characterization of fusion-positive rhabdomyosarcoma genomics and ctDNA detection and quantification. (A) CNAs were identified in 25 of 35 samples (71%) by ULP-WGS. (B) Translocations (rearrangement) were detected in 28 of 30 samples (93%) with sufficient sequencing library available for sequencing by Rhabdo-Seq. Of 35 tumors, five did not have sufficient DNA material quality for Rhabdo-Seq panel sequencing. (C) Sixteen percent of serum samples had detectable ctDNA by ULP-WGS. (D) ctDNA was detected by identifying the PAX/FOXO1 translocation in 55% of serum samples. (E) In total, 57% of patients had detectable ctDNA using both ULP-WGS for CNA detection and Rhabdo-Seq panel for gene rearrangement detection. (F) Plotted are the percent ctDNA values for fusion-negative rhabdomyosarcoma samples with detectable ctDNA by ULP-WGS (blue circles) and by Rhabdo-Seq (red circles). ctDNA content estimates are similar in most cases where ctDNA is detected by both methods. However, the lack of aneuploidy in some cases results in ctDNA being unmeasurable by ULP-WGS. (G) Absolute copy numbers of PAX3, PAX7, and FOXO1 locus were inferred from the gene locus using ULP-WGS data from tumor samples. In all the PAX7/FOXO1 tumors available, both partner genes were amplified from 5 to 18 times explaining the overestimate of ctDNA content in three cases with detectable ctDNA by PAX7/FOXO1 detection. CNA, copy number alteration; ctDNA, circulating tumor DNA; ULP-WGS, ultralow passage whole-genome sequencing.

FIG 3.

(A) EFS and (B) OS by ctDNA detection status, by ULP-WGS for FN-RMS, and translocation detection for FP-RMS of the entire cohort. (C) EFS and (D) OS by ctDNA detection status, determined by ULP-WGS for FN-RMS and translocation detection for FP-RMS, of the entire cohort excluding cases with MYOD1^L122R mutations. (E) EFS and (F) OS by ctDNA status, determined by ULP-WGS, in patients with FN-RMS after excluding cases with MYOD1^L122R mutations. (G) EFS and (H) OS by ctDNA status, determined by translocation detection, in patients with FP-RMS. P value from the log-rank test. ctDNA, circulating tumor DNA; EFS, event-free survival; FN-RMS, fusion-negative rhabdomyosarcoma; FP-RMS, fusion-positive rhabdomyosarcoma; OS, overall survival; RMS, rhabdomyosarcoma; ULP-WGS, ultralow passage whole-genome sequencing.